This year we are again participating in the many projects relating to the analysis of peptides and proteins that have been brought to our attention by researchers at NHLBI and NIH. The goal is still to use advanced mass spectrometric technology to study the biology of a particular system by obtaining information on the identity of proteins either through mass spectrometric MS/MS sequencing or by simply carefully measuring the masses of the protein itself. This technique is unique in identifying post-translational modifications (PTMs) essential for protein/cellular functions. With the state-of-the-art Micromass QTOF3 mass spectrometer and other LC-MS and MALDI-TOF instruments, we are able to perform many experiments on biological samples, mostly in this protein and peptide class. In addition,we seek new chemical methods to enhance the search routines in order to increase their reliability. Currently, we are studying the proteomics of lung and Chinese hamster ovary cells, identifying tumor susceptibility proteins, and determining their modifications. New this year are studies of PTMs of mitochondrial electron transport chain complexes essential for energy production in the cell. We have identified several novel phosphorylation sites in rat ATP synthase F1 complex beta subunit and we are currently studying glycosylation and oxidation in mitochondrial proteins using 2D gel electrophoresis and mass spectrometry (Balaban). We have recently looked at human papillomavirus in an effort to determine the linking of its disulfide bridges by using specialized bridge-locating software (Buck). A series of extracts from Camellia sinensis separated by countercurrent precipitation chromatography were exmined for the agent responsible for carotenoid cleavage enzymes(Ito). A series of gel spots derived from stem cells from skeletal muscle that turns into brain tissue when cultured were examined for evidence of glycosylation (Epstein). We have tentatively identified myosin heavy chain IIA as the antigen for a class of CLL antibodies (Chu).[unreadable] We continue to spend a significant amount of time optimizing the sensitivity and resolution of our QTOF3,its capillary LC and its communication with the mass spectrometer, in particular with regard to its ability to analyze proteins as opposed to peptides.